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Of up to 300,000,000 or more human sperm that can be ejaculated during coitus, only about 200 reach the site of fertilization in the oviduct before fertilisation occurs. Once a sperm finds an egg, it must first migrate through the layers of granulosa cells that surround the egg and then bind to and cross the zona pellucida. Finally, it must bind to and fuse with the egg plasma membrane.
The human sperm must first be modified by conditions in the female reproductive tract. Sperm penetration of ZP and the later events of fertilization require acrosomal exocytosis. Successful fertilization depends heavily on the activation of all the components of functional competence, namely, forward motility, hyperactivated motility, and acrosomal exocytosis.
The sperm undergo biochemical and functional changes, including changes in glycoproteins, lipids, and ion channels in the sperm plasma membrane and a large change in the resting potential of this membrane (the membrane potential moves to a more negative value so that the membrane becomes hyperpolarized).
Contraceptives usage is on the rise in our society as the world modernizes. With more information given to the community, couples can now choose the appropriate and suitable contraceptive in birth control or disease-preventing. The molecular events of the sperm prior to and during fertilization will be examined and some examples of contraceptives that target these processes.
Contraceptive use has a broad range of benefits to the women, men, their families and communities. Unplanned pregnancies usually results in abortions that lead to emotional and physical trauma to both men and women, usually more on the maternal side. In order to have healthier pregnancies, desirable family size and sufficient time interval between children, couples should adopt the use of contraceptives. Some contraceptives are also used in disease preventing, such as the condom. There are four types of contraception: natural, hormonal, mechanical and surgical.
A natural contraceptive method such as the calendar planning is the avoidance of sexual intercourse during the ovulation phase. Hormonal contraceptives include the pill or the skin patch which usually releases estrogen and progestin hormones to prevent ovulation, thicken the vaginal mucus and is highly effective. Condoms, spermicides, diaphragms are some examples of mechanical contraception. Surgical contraception involves either the female or male sterilization. In females, there is tubal ligation and in males, there is vasectomy to cut the vas deferens.
Mammalian fertilization normally begins when a sperm, which has undergone
capacitation in the female genital tract, binds to the zona pellucida surrounding an egg in the ampulla region of the oviduct. This induces an acrosome reaction in the sperm, releasing the contents of the acrosomal vesicle, which contains enzymes that help the sperm to digest a passage through the zona. The acrosome reaction is also required for the sperm to bind to and fuse with the egg plasma membrane.
The fusion of the sperm with the egg induces a Ca2+ wave and oscillations in the egg cytosol, which activate the egg. The activation includes the egg cortical reaction, in which cortical granules release their contents, which alter the zona pellucida so that other sperm cannot bind to or penetrate it. The Ca2+ signal also triggers the development of the zygote, which begins after the two haploid pronuclei have come together and their chromosomes have aligned on a single mitotic spindle, which mediates the first mitotic division of the zygote.
The mature spermatozoon consists of three distinct regions: the head, the middle piece and the tail.
Figure 1: Sperm anatomy. The head consists principally of a nucleus, which contains highly condensed chromatin. The acrosomal cap is located at the tip of the head. This is a modified lysosome that contains hydrolytic enzymes required for helping the sperm to penetrate the egg's zona pellucida. When a sperm contacts the egg coat, the contents of the vesicle are released by exocytosis in the acrosome reaction. This reaction is required for the sperm to bind to the coat, burrow through it, and fuses with the egg.
A short neck joins the head to the middle piece and contains the centrioles of the original spermatid. Mitochondria are also found arranged in a spiral around the microtubules. Mitochondrial activity is essential for production of ATP for motility of the tail.
The motile tail appears as a whip-like organelle. The long flagellum of the spermatozoon has a complex and corkscrew motion. The axoneme consists of two central singlet microtubules surrounded by nine evenly spaced microtubule doublets. The dense fibers are stiff and non-contractile, and they are thought to restrict the flexibility of the flagellum and protect it from shear forces. The active bending of the flagellum is caused by the sliding of adjacent microtubule doublets past one another, driven by dynein motor proteins, which use the energy of ATP hydrolysis to slide the microtubules.
A mature spermatozoon lacks endoplasmic reticulum, golgi apparatus, lysosomes, peroxisomes, inclusions and many other intracellular structures so as to reduce the cell's size and mass. It is essentially a mobile carrier for the enclosed chromosomes and extra weight will slow it down. As the cell lacks glycogen or other energy reserves, it must absorb nutrients (primarily fructose) from the surrounding fluid.
Mature male gametes, also known as sperm or spermatozoa, are produced via the process of spermatogenesis. The process begins when the male reaches puberty and occurs continuously in the seminiferous tubules.
Stem cells, also known as spermatogonia, divide by mitosis to produce two daughter cells, one of which remains at that location as a spermatogonium while the other differentiates into a primary spermatocyte.
Meiosis takes place and forms the secondary spermatocytes, which differentiate further into spermatids. Spermatids are immature gametes that differentiate into spermatozoa through the process of spermiogenesis. At spermiation, a spermatozoon loses its attachment to the Sertoli cell and enters the lumen of the seminiferous tubules. The entire process, from the spermatogonial division to spermiation, takes approximately ten weeks.
Composition of semen
There are approximately 20 to 100 million spermatozoa per ml of semen. A mixture of glandular secretions with a distinct ion and nutrient composition makes up the seminal fluid. Sixty percent is from the seminal vesicles, 20-30% from the prostate glands, 5% Sertoli cells and epididymis and less than 5% from bulbourethral glands. Enzymatic materials like proteases, seminal plasmin, prostatic enzymes and fibrinolysin are also found in the semen.
The first step of capacitation includes biochemical events such as stimulation of soluble adenylyl cyclase activity, protein nitrosylation and protein tyrosine phosphorylation. Capacitation also includes the second and third phases of activation: initiation of hyperactivated motility and the acquisition of competence for acrosomal exocytosis. Capacitation takes about 5-6 hours in humans and is completed only when the sperm arrive in the oviduct.
Competence of a sperm to participate in fertilisation depends upon certain key functions including sperm motility and acrosomal exocytosis. Ejaculated mammalian sperm are initially not competent to accomplish any of these tasks: competence involves maturation at specific sites and at particular times to enable them to become competent for fertilization. Inactive spermatozoa that are deposited into the vagina become activated. They display forward motility and acquire hyperactivated motility characterised by assymetrical flagellar beats with increased amplitude of the flagellar bend.
Hyperactivation requires the alkalinization of the sperm and is calcium-dependent. Calcium is mobilized into the sperm from the external environment through plasma membranes channels: for example, members of the CATSPER (cation channel, sperm associated) family, and is also released internally from intracellular stores, such as the nuclear envelope located at the base of the sperm flagellum or the acrosome.
Following intracellular alkalinization, the membrane potential is altered, producing a rapid hyperpolarization of the cell. This hyperpolariztion is caused by a weak outwardly rectifying K+ current (IKSper) originating from the principal piece of the sperm flagellum. Alkalinization activates the pHi-sensitive IKSper, creating a negative membrane potential where Ca2+ antry via ICATSPER is maximised.
The proton channel Hv1 has recently been identified as the predominant effector of proton extrusion and is located in the flagellum. This outward transport of protons is activated by membrane depolarization, and is dependent on an alkaline extracellular environment. Sperm also relies on Na, K-ATPase α4 for fertility. The sperm sodium-hydrogen exchanger (SLC9A10) is a cation-proton anti-porter that may participate in the alkalinization of sperm during capacitation. It is essential in the sperm's motility.
Another essential in sperm capacitation is the chloride channels. The absence of Cl- does not affect sperm viability, but processes such as an increase in protein-tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, ZP-induced acrosomal exocytosis, and fertilization are abolished or significantly reduced when chloride is replaced by gluconate.
Capacitated Sperm Bind to the Zona Pellucida and Undergo an Acrosome Reaction
During ovulation, mammalian eggs are released from the ovary into the peritoneal cavity next to the entrance to the oviduct, into which they are rapidly swept. They are covered with several layers of granulosa cells embedded in an extracellular matrix that is rich in hyaluronic acid. The granulosa cells may help the egg get picked up into the oviduct, and they may also secrete unidentified chemical signals that attract sperm to the egg.
Figure 2: Fertilization process. Adapted from Inoue, N., et al., 2005.
On encountering an egg, a capacitated sperm first must penetrate the layers of granulosa cells, making use of a hyaluronidase enzyme on the surface of the sperm. It can then bind to the zona pellucida. The zona usually acts as a barrier to fertilization across species, and removing it often eliminates this barrier.
The zona pellucida of most mammalian eggs is composed mainly of three glycoproteins, all of which are produced exclusively by the growing oocyte. Two of them, ZP2 and ZP3, assemble into long filaments, while the other, ZP1, crosslinks the filaments into a three-dimensional network. The ZP3 protein is crucial: female mice with a disrupted Zp3 gene produce eggs that lack a zona and are infertile. O-linked oligosaccharides on ZP3 seem to be at least partly responsible for the species-specific binding of sperm to the zona. The binding of sperm to the zona is complex, however, and involves both ZP3-dependent and ZP3-independent mechanisms and a variety of proteins on the sperm surface.
Acrosomal exocytosis occurs as a result of stimulation by ZP proteins or progesterone. The acrosome reaction is required for normal fertilization, as it exposes various hydrolytic enzymes that are believed to help the sperm tunnel through the zona pellucida, and it alters the sperm surface so that it can bind to and fuse with the plasma membrane of the egg.
Four stages of exocytosis represent: acrosomes that are intact; acrosomes that have exposed internal proteins (ZP3R/sp56) but have not released soluble components (EGFP); acrosomes that have released soluble EGFP but still retain acrosomal matrix ZP3R; and acrosomes that have lost both the soluble EGFP and matrix ZP3R.
In vitro, purified ZP3 can trigger the acrosome reaction, possibly by activating a lectin-like receptor on the sperm surface, thought to be a transmembrane form of the enzyme galactosyltransferase. Receptor activation leads to an increase in Ca2+ in the sperm cytosol, which initiates the exocytosis.
During capacitation, acrosomal swelling causes changes in distance between the outer acrosomal membrane and the plasma membrane, and it is likely that contact between these two membranes establishes acrosome docking or the formation of fusion pores. The formation of these intermediate stages is coincident with stabilization of a primed event (membrane docking/ fusion) so that the exocytosis is restricted and occurs more rapidly in response to a specific stimulus (e.g. ZP or progesterone).
The actin cytoskeleton is important regulator of acrosomal exocytosis. Actin has been localized on the external surface of the fibrous sheath of human sperm. Actin is present in its monomeric, globular form (G-actin) as well as filamentous actin (F-actin). During capacitation, G-actin is polymerized to F-actin. Inhibition of actin depolarization thjat phalloidin in a permeabilized sperm model inhibits acrosomal exocytosis, indicating that the dispersion of F-actin is necessary for acrosomal exocytosis to occur. Thus, F-actin stabilizes the exocytotic machinery to restrict secretory granules from fusing with the plasma membrane. When capacitated sperm are stimulated to undergo acrosomal exocytosis, rapid F-actin depolymerization occurs. Moreover, inhibition of actin polymerization blocks ZP-induced acrosomal exocytosis and sperm penetration into ZP-free eggs, thus interfereing with the ability of sperm to become competent for in vitro fertilization.
A rise in intracellular calcium also plays a role in depolymerization of actin, resulting in acrosomal exocytosis. One theory states that the acrosome is a calcium store and that depletion of Ca2+ from the acrosome activates store-operated channels that allow sustained entry of Ca2+ from the medium. On the other hand, others have shown that the Ca2+ rise induced by exposure to ZP or progesterone starts at different sites within the sperm head, indicating that these agonists may stimulate acrosomal exocytosis via different, stimulus-specific mechanisms.
After a sperm has undergone the acrosome reaction and penetrated the zona
pellucida, it binds to the egg plasma membrane at the tips of the microvilli on the egg surface. The sperm binds initially by its own tip and then by its side. Neighboring microvilli on the egg surface rapidly elongate and cluster around the sperm to ensure that it is held firmly so that it can fuse with the egg. After fusion, all of the sperm contents are drawn into the egg, as the microvilli are resorbed. The molecular mechanisms responsible for sperm-egg binding and fusion are largely unknown, although two membrane proteins have been shown to be required for the fusion. One is a sperm-specific transmembrane protein of the immunoglobulin superfamily called Izumo. It becomes exposed on the surface of mouse and human sperm during the acrosome reaction. Anti-Izumo antibodies block the fusion, and Izumo-deficient mouse sperm fail to fuse with normal eggs, but it is still unknown how Izumo promotes sperm-egg fusion. The only protein on the egg surface demonstrated to be required for fusion with a sperm is the CD9 protein, which is a member of the tetraspanin family, so-called because these proteins have four membrane-spanning segments. Normal sperm fail to fuse with CD9-deficient mouse eggs, indicating that sperm-egg fusion depends on CD9, but it is not known how. CD9 does not act alone on the egg surface to promote fusion: normal sperm also fail to fuse with eggs treated 5 mm with an enzyme that removes proteins attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor, indicating that one or more GPI-linked proteins is also required for fusion, although the relevant protein or proteins have yet to be identified.
Sperm Fusion Activates the Egg by Increasing Ca2+ in the Cytosol
Fusion with a sperm activates the egg, causing the cortical granules to release their contents by exocytosis, a process called the cortical reaction. Meiosis, which was arrested in metaphase II, resumes, producing a second polar body, and a zygote, which begins to develop. An increase in Ca2+ in the cytosol of the fertilized egg triggers all of these events.
When the sperm fuses with the egg plasma membrane in the normal way, it causes a local increase in cytosolic Ca2+, which spreads through the cell in a wave. The wave propagates by positive feedback: the increase in cytosolic Ca2+ causes Ca2+ channels to open, allowing still more Ca2+ to enter the cytosol. The initial wave of Ca2+ release is usually followed within a few minutes by Ca2+ oscillations, which persist for several hours. The fused sperm triggers the Ca2+ wave and oscillations by bringing a factor into the egg cytosol. All of these treatments increase the concentration of inositol 1,4,5-trisphosphate (IP3), which releases Ca2+ from the endoplasmic reticulum and initiates the Ca2+ wave and oscillations. A strong candidate for the critical factor that mammalian sperm introduce into the egg is a sperm-specific form of phospholipase C (PLCz), which directly cleaves phosphoinositol 4,5-bisphosphate (PI(4,5)P2) to produce IP3 (and diacyglycerol).
The Cortical Reaction Helps Ensure That Only One Sperm Fertilizes the Egg
Although many sperm can bind to an egg, normally only one fuses with the egg plasma membrane and injects its cytosol, nucleus, and other organelles into the egg cytoplasm. If more than one sperm fuses-a condition called polyspermy- extra or multipolar mitotic spindles are formed, resulting in faulty segregation of chromosomes during the first mitotic cell divisions; aneuploid cells are produced, and development usually stops.
Two mechanisms operate to ensure that only one sperm fertilizes the egg.
First, a change in the egg plasma membrane caused by the fusion of the first sperm prevents other sperm from fusing. In mammalian eggs, the mechanism is not known. The second block to polyspermy is provided by the egg cortical reaction, which releases various enzymes that change the structure of the zona pellucida so that sperm cannot bind to or penetrate it. Among these changes is the inactivation of ZP3 so that it can no longer bind sperm or induce an acrosome reaction; in addition, ZP2 is cleaved, which also helps to make the zona impenetrable.
Potential targets of contraceptives
On one hand, Ca+ increases the motility in the epidymal sperm, and on the other hand, an elevated intracellular Ca+ in the ejaculated sperm induces sperm death. [Gupta, A. et al, 2005.] An increased extracellular concentration of Ca+ does not appear to inï¬‚uence the motility of ejaculated spermatozoa, suggesting a well-developed system for Ca+ inï¬‚ux. However, decreasing the extracellular Ca+ concentration (below 65%) results in total inhibition of sperm motility. Ca+ also indirectly inï¬‚uences cAMP and pH of the spermatozoa via other cellular mechanisms that are known to be important regulatory factors of sperm motility, thus playing a pivotal role in sperm function. Intrasperm calcium accumulation caused by drugs introduced to ejaculated sperm have shown to produce spermicidal action and can be used as contact spermicides [Reddy et al., 2002].
An example will be the Nonoxynol-9 which is the most widely used contact spermicide, a non-ionic sufactant. [Gupta, A. et al, 2005.] Nonoxynol-9 is commercially available as pessary, foam, gel, cream formulations in various strengths. N-9 targets the spermatozoa's cell membrane at its neck. Soon after contact, the plasma membrane and acrosomal membrane is destroyed. The midpiece membrane, mitochondrial cristae is also absent. Evidence of vesiculations is shown by all the damage done to the membranes which cause them to be loose and detached. This irreversible membrane alterations cause an immediate devitalization of the spermatozoa, causing loss of function, motility.